Pathogenesis of cardiomyopathy

ABSTRACT

Disclosed within is a mouse, and cells derived therefrom, which are homozygous for a disrupted δ-sarcoglycan gene, the disruption in said gene having been introduced into the mouse or an ancestor of the mouse at an embryonic stage. Said disruption prevents the synthesis of functional δ-sarcoglycan in cells of the mouse and results in the mouse having a reduced amount of β- and ε-sarcoglycan and sarcospan, and a disruption of the sarcoglycan-sarcospan complex in smooth muscle of the mouse. Said disruption also results in a reduced amount of sarcospan, α-, β-, γ-, and ε-sarcoglycan in the sarcolemma of skeletal and cardiac muscles of the mouse, compared to the amounts of said components in a mouse lacking disrupted δ-sarcoglycan genes. Preferred specific disruptions of the δ-sarcoglycan gene are listed. Also disclosed is a mouse, and cells derived therefrom, which are homozygous for a disrupted β-sarcoglycan gene, the disruption in said gene having been introduced into the mouse or an ancestor of the mouse at an embryonic stage. The disruption prevents the synthesis of functional β-sarcoglycan in cells of the mouse and results in the mouse having a reduced amount of δ- and ε-sarcoglycan and sarcospan and α-dystroglycan in smooth muscle of the mouse. The disruption also results in a disruption of the sarcoglycan-sarcospan complex in smooth muscle of the mouse, and a reduced amount of sarcospan, α, γ, δ- and ε-sarcoglycan in the sarcolemma of skeletal and cardiac muscles of the mouse, compared to the amounts of the components in a mouse lacking disrupted β-sarcoglycan genes. Preferred specific disruptions of the β-sarcoglycan gene are listed. A method for treating mammalian autosomal recessive limb-girdle muscular dystrophy type 2F in an individual is also disclosed. The method comprises, providing an expression vector which encodes a wild-type form of δ-sarcoglycan, and introducing the expression vector into skeletal and smooth muscle tissue of the individual under conditions appropriate for expression of the wild-type form of δ-sarcoglycan in said tissues. Examples of expression vectors for use in this method are adenovirus expression vector, a gutted adenovirus expression vector, and an adeno-associated expression vector. Also disclosed are methods for treating mammalian autosomal recessive limb-girdle muscular dystrophy type 2E, and type 2F, in an individual. The methods comprise, providing an expression vector which encodes a wild-type form of β-sarcoglycan, or δ-sarcoglycan, respectively, and introducing the expression vector into skeletal and smooth muscle tissue of the individual under conditions appropriate for expression of the wild-type form of the sarcoglycan gene in said tissues. The δ-sarcoglycan deficient, and β-sarcoglycan deficient mice of the present invention are useful in identifying therapeutic compounds for treatment of an individual diagnosed with δ-sarcoglycan-deficient limb-girdle muscular dystrophy, and β-sarcoglycan-deficient limb-girdle muscular dystrophy, respectively. A therapeutic method for treating ischemic heart disease caused by reduced expression of the sarcoglycan-sarcospan complex in vascular smooth muscle cells of an individual is also provided. The method comprises contacting the vascular smooth muscle cells of the individual with a vascular smooth muscle relaxant, such as Nicorandil. This method is also useful for preventing ischemic injury in skeletal and cardiac muscle of an individual caused by reduced expression of the sarcoglycan-sarcospan complex in the vascular smooth muscle cells of the individual. The method is also useful for treating mammalian autosomal recessive limb-girdle muscular dystrophy type 2F or type 2E in an individual. Other methods provided include a method for identifying a therapeutic compound for the treatment of ischemic heart disease in an individual caused by reduced expression of the sarcoglycan-sarcospan complex in the vascular smooth muscle cells of the individual, and also a method for identifying a therapeutic compound for the prevention of ischemic injury in skeletal and cardiac muscle of an individual which is caused by reduced expression of the sarcoglycan-sarcospan complex in vascular smooth muscle cells of the individual.

BACKGROUND OF THE INVENTION

[0001] The sarcoglycan complex is a group of single pass transmembraneproteins (α, β, δand γ-sarcoglycan) which is tightly associated withsarcospan to form a subcomplex within the dystrophin-glycoproteincomplex (DGC) in skeletal and cardiac muscle (Campbell et al., Nature338: 259-362 (1989); Yoshida et al., J. Biochem. 108: 748-752 (1990);Crosbie et al., J. Cell Biol. 145: 153-165 (1999)). The DGC is furthercomprised of dystrophin, the dystroglycan complex and the syntrophins(Hoffman et al., Cell 51: 919-928 (1987); Froehner et al., Soc. Gen.Physiol. Ser. 52: 197-207 (1997); Durbeej et al., Curr. Opin. Cell.Biol. 10: 594-601 (1998)). The expression of the sarcoglycan-sarcospancomplex is necessary to target dystroglycan to the sarcolemma (Duclos etal., J. Cell Biol. 142: 1461 -1471 (1998); Duclos et al., Neuromusc.Disord. 8: 30-38 (1998); Holt et al., Mol. Cell 1: 841-848 (1998);Straub et al., Am. J. Path. 153: 1623-1630 (1998)) which in turn confersa link between the extracellular matrix and the F-actin cytoskeleton(Ervasti et al., J. Cell Biol. 122: 809-823 (1993)). Thus, the DGC isthought to protect muscle cells from contraction-induced damage (Petrofet al., Proc. Natl. Acad. Sci. USA 90: 3710-3714 (1993)). In agreementwith this hypothesis, mutations in the genes for the sarcoglycans,dystrophin and laminin α2 chain are responsible for limb-girdle musculardystrophy, Duchenne/Becker muscular dystrophy and congenital musculardystrophy respectively (Straub et al., Curr. Opin. Neurol. 10: 168-175(1997); Lim et al., Curr. Opin. Neurol. 11: 443-452 (1998)). Clinicalevidence of cardiomyopathy is variably present in these musculardystrophies (Towbin, J. A., Curr. Opin. Cell Biol. 10: 131-139 (1998))but a correlation between the primary mutation of the sarcoglycan genesand cardiomyopathy is yet to be established (Melacini et al., Muscle &Nerve 22: 473-479 (1999)).

[0002] Dilated cardiomyopathy is a multifactorial disease that includesboth inherited and acquired forms of cardiomyopathy. Inheritedcardiomyopathy in humans can be associated with genetic defectsoccurring in components of the dystrophin-glycoprotein complex (DGC)(Towbin, J. A., Curr. Opin. Cell Biol. 10: 131-139 (1998)). Mutations inthe dystrophin gene lead to a high incidence of cardiomyopathy inDuchenne and Becker muscular dystrophy patients (DMD/BMD) and can causeX-linked dilated cardiomyopathy (Towbin, J. A., Curr. Opin. Cell Biol.10: 131-139 (1998)). In addition to these primary genetic causes ofcardiomyopathy, recent data suggest that disruption of the DGC underliethe cardiomyopathy associated with enteroviral infection (Badorff etal., Nat. Med. 5: 320-326 (1999)). Consequently, evidence isaccumulating that the DGC plays a critical role in the pathogenesis ofsome forms of inherited and acquired cardiomyopathy. Several componentsof the DGC are also expressed in smooth muscle (Houzelstein et al., J.Cell Biol. 119: 811-821 (1992); North et al., J. Cell Biol. 120:1159-1167 (1993); Ozawa, et al., Hum. Mol. Gen. 4: 1711-1716 (1995);Durbeej et al., Curr. Opin. Cell. Biol. 10: 594-601 (1998)).Interestingly, potential smooth muscle dysfunction has been described inpatients with Duchenne muscular dystrophy (Bahron et al., N. Engl. J.Med. 319: 15-18 (1998); Jaffe et al., Arch. Phys. Med. Rehabil. 71:742-744 (1990)). However, no smooth muscle dysfunction has been reportedin patients with limb-girdle muscular dystrophy.

[0003] Recently, a fifth sarcoglycan, ε-sarcoglycan, was cloned andshown to be highly homologous to α-sarcoglycan (Ettinger et al., J.Biol. Chem. 272: 32534-32538 (1997); McNally et al., FEBS Lett. 422:27-32 (1998)). ε-sarcoglycan is expressed in skeletal and cardiacmuscle, but also in several non-muscle tissues. Whether ε-sarcoglycan isassociated with the other sarcoglycans in striated muscle is yet to bedetermined. At the immunofluorescence level, however, it has been shownthat ε-sarcoglycan is still present in skeletal muscle of α-sarcoglycandeficient (Sgca-null mice) mice although the other sarcoglycans aregreatly reduced (Duclos et al., J. Cell Biol. 142: 1461-1471 (1998)).This indicates that ε-sarcoglycan is not an additional member of theknown tetrameric complex of α-, β-, γ- and δ-sarcoglycan in skeletalmuscle but may be part of a distinct complex at the sarcolemma.

[0004] Sgca-null mice have recently been reported to display aprogressive muscular dystrophy (Duclos et al., J. Cell Biol. 142:1461-1471 (1998)). The primary absence of α-sarcoglycan was accompaniedby the concomitant loss of β-, γ- and δ-sarcoglycan and sarcospan inskeletal and cardiac muscle fibers, a phenomenon that is also observedin human forms of sarcoglycanopathies (Lim et al., Curr. Opin. Neurol.11: 443-52 (1998)). Interestingly, although the SG-SSPN complex wasabsent from the cardiac muscle membrane, no morphological signs ofcardiomyopathy were observed (Duclos et al., J. Cell Biol. 142:1461-1471 (1998)).

SUMMARY OF THE INVENTION

[0005] One aspect of the present invention relates to a mouse, and cellsderived therefrom, which is homozygous for a disrupted δ-sarcoglycangene, the disruption in said gene having been introduced into the mouseor an ancestor of the mouse at an embryonic stage. Said disruptionprevents the synthesis of functional δ-sarcoglycan in cells of the mouseand results in the mouse having a reduced amount of β- and ε-sarcoglycanand sarcospan, and a disruption of the sarcoglycan-sarcospan complex insmooth muscle of the mouse. Said disruption also results in a reducedamount of sarcospan, α-, β-, γ-, and ε-sarcoglycan in the sarcolemma ofskeletal and cardiac muscles of the mouse, compared to the amounts ofsaid components in a mouse lacking disrupted δ-sarcoglycan genes.Preferred specific disruptions of the δ-sarcoglycan gene are listed.

[0006] Another aspect of the present invention relates to a mouse, andcells derived therefrom, which is homozygous for a disruptedβ-sarcoglycan gene, the disruption in said gene having been introducedinto the mouse or an ancestor of the mouse at an embryonic stage. Thedisruption prevents the synthesis of functional β-sarcoglycan in cellsof the mouse and results in the mouse having a reduced amount of δ- andε-sarcoglycan and sarcospan and α-dystroglycan in smooth muscle of themouse. The disruption also results in a disruption of thesarcoglycan-sarcospan complex in smooth muscle of the mouse, and areduced amount of sarcospan, α-, γ- , δ- and ε-sarcoglycan in thesarcolemma of skeletal and cardiac muscles of the mouse, compared to theamounts of the components in a mouse lacking disrupted β-sarcoglycangenes. Preferred specific disruptions of the β-sarcoglycan gene arelisted.

[0007] Another aspect of the present invention is a method for treatingmammalian autosomal recessive limb-girdle muscular dystrophy type 2F inan individual. The method comprises, providing an expression vectorwhich encodes a wild-type form of δ-sarcoglycan, and introducing theexpression vector into skeletal and smooth muscle tissue of theindividual under conditions appropriate for expression of the wild-typeform of δ-sarcoglycan in said tissues. Examples of expression vectorsfor use in this method are adenovirus expression vector, a guttedadenovirus expression vector, and an adeno-associated expression vector.In one embodiment, the expression vector contains a muscletissue-specific promoter. One method of introduction into the skeletalmuscle is by intramuscular injection.

[0008] Another aspect of the present invention is a method for treatingmammalian autosomal recessive limb-girdle muscular dystrophy type 2E inan individual. The method comprises, providing an expression vectorwhich encodes a wild-type form of β-sarcoglycan, and introducing theexpression vector into skeletal and smooth muscle tissue of theindividual under conditions appropriate for expression of the wild-typeform of β-sarcoglycan in said tissues. Examples of expression vectorsfor use in this method are adenovirus expression vector, a guttedadenovirus expression vector, and an adeno-associated expression vector.In one embodiment, the expression vector contains a muscletissue-specific promoter. One method of introduction into the skeletalmuscle is by intramuscular injection.

[0009] The δ-sarcoglycan deficient, and β-sarcoglycan deficient mice ofthe present invention are useful in identifying therapeutic compoundsfor treatment of an individual diagnosed with δ-sarcoglycan-deficientlimb-girdle muscular dystrophy, and β-sarcoglycan-deficient limb-girdlemuscular dystrophy, respectively.

[0010] Another aspect of the present invention is a therapeutic methodfor treating ischemic heart disease caused by reduced expression of thesarcoglycan-sarcospan complex in vascular smooth muscle cells of anindividual. The method comprises contacting the vascular smooth musclecells of the individual with a vascular smooth muscle relaxant, such asNicorandil. Such reduced expression of the sarcoglycan-sarcospan complexin vascular smooth muscle cells of the individual may be due to a defectin the δ-sarcoglycan genes of the individual, or to a defect in theβ-sarcoglycan genes of the individual. This method is also useful forpreventing ischemic injury in skeletal and cardiac muscle of anindividual caused by reduced expression of the sarcoglycan-sarcospancomplex in the vascular smooth muscle cells of the individual. Themethod is also useful for treating mammalian autosomal recessivelimb-girdle muscular dystrophy type 2F or type 2E in an individual.Other methods provided include methods for identifying a therapeuticcompound for the treatment of ischemic heart disease in an individualcaused by reduced expression of the sarcoglycan-sarcospan complex in thevascular smooth muscle cells of the individual, and also method foridentifying a therapeutic compound for the prevention of ischemic injuryin skeletal and cardiac muscle of an individual which is caused byreduced expression of the sarcoglycan-sarcospan complex in vascularsmooth muscle cells of the individual.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 is a schematic representation of targeted disruption of themouse Sgcd-gene. Shown is a restriction map of the 5′ portion of theSgcd-gene showing wild type allele (top), the targeting vector (middle),and the predicted targeted allele following homologous recombination(bottom). The position of the neo and tk cassettes, hybridization sitesof probes A and B, for Southern analyses, and the primers used forPCR-based genotyping (→) are indicated.

[0012]FIG. 2 is a schematic representation of targeted disruption of themouse Sgcb-null mutant mice. Shown is a restriction map of the wild-typeSgcb allele (top), the targeting vector (middle), and the predictedtargeted allele following homologous recombination (bottom). Exons 3, 4,5 and 6 were replaced by phosphoglycerate kinase neomycin cassette(neo). The position of the neo and tk cassettes, hybridization sites ofprobes 1 and 2, for Northern and Southern analyses, are also indicated.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Aspects of the present invention are based upon the generationand study of Sgcd-null mice (δ-sarcoglycan knockout), and Sgcb-null(β-sarcoglycan knockout) mice. The homozygous disruption of either ofthese genes in the mouse results in an absence of thesarcoglycan-sarcospan (SG-SSPN) complex in skeletal and cardiacmembranes and also the loss of a newly identified unique vascular smoothmuscle SG-SSPN complex. Surprisingly, the disruption of the SG-SSPNcomplex in vascular smooth muscle of the mice perturbs vascularfunction, which initiates cardiomyopathy and exacerbates musculardystrophy.

[0014] Cardiomyopathy is a common clinical phenotype of patients withcertain forms of limb-girdle muscular dystrophy. Cardiac involvement hasbeen previously documented in patients with primary mutations of β-, γ-and δ-sarcoglycan (Moreira et al., J. Med. Genet. 35: 951-953 (1998);Melacini et al., Muscle Nerve 22: 473-479 (1999), RD Cohn and T. Voit,personal communication) while patients with mutations in theα-sarcoglycan gene (the most common form of sarcoglycanopathies) onlyrarely display mild forms of cardiomyopathy (Melacini et al., MuscleNerve 22: 473-479 (1999)). However, prior to this disclosure, theinvolvement of the specific sarcoglycans in the pathogenesis ofcardiomyopathy and muscular dystrophy, with respect to tissuedistribution, was largely unknown.

[0015] One aspect of the present invention is a mouse, and cells derivedtherefrom, which is homozygous for a disrupted δ-sarcoglycan gene. Thedisruption in the δ-sarcoglycan gene is introduced into the mouse or anancestor of the mouse at an embryonic stage. The introduced disruptionnecessarily prevents the synthesis of functional δ-sarcoglycan in allcells of the mouse. The molecular consequences of the mutation in themouse includes, without limitation, a reduction in the amount ofsarcospan, β- and ε-sarcoglycan in smooth muscle, and also a disruptionof the sarcoglycan-sarcospan complex in smooth muscle. In addition,there is a reduced amount of sarcospan, α-, β-, γ-, and ε-sarcoglycan inthe sarcolemma of skeletal and cardiac muscle of the mouse. Thereduction in the amount of the aforementioned molecules are determinedby comparison to the amounts of these molecules in the correspondingtissue of a mouse lacking a disrupted δ-sarcoglycan gene.

[0016] One of skill in the art will recognize that functional disruptionof the δ-sarcoglycan gene can be achieved by several approaches.Generally, a specific disruption of the δ-sarcoglycan gene is made in aprogenitor cell (embryonic stem cells) of the mouse. A mouse which isheterozygous for the disrupted gene is produced, and a mouse homozygousfor the disrupted gene is produced by mating two heterozygotes. In oneembodiment, the disruption in the δ-sarcoglycan gene results in adeletion of a region of 2992 base pairs, the region including 2277 basepairs of intron 1, the entire exon 2, and 576 base pairs of intron 2.This deleted region of the mouse genome is replaced with a PGK-neomycincassette as a marker for neomycin resistance. Such a disruption can beproduced by introduction, into embryonic stem cells, of a DNA constructwhich has 5 kb of intron 1 and 5 kb of intron 2 of the wild typeδ-sarcoglycan gene, but lacks exon 2, and instead has a neomycinresistance gene inserted between the sequences from intron 1 and intron2 sequences, in the opposite transcriptional orientation as the exon 2sequences which it is replacing.

[0017] Another aspect of the present invention is a mouse, and cellsderived therefrom, which is homozygous for a disrupted β-sarcoglycangene. The disruption in the β-sarcoglycan gene having been introducedinto the mouse or an ancestor of the mouse at an embryonic stage. Thedisruption necessarily prevents the synthesis of functionalβ-sarcoglycan in all cells of the mouse. The molecular consequences ofthe mutation in the mouse includes, without limitation, a reduction inthe amount of sarcospan, δ- and ε-sarcoglycan in smooth muscle, and alsoa disruption of the sarcoglycan-sarcospan complex in smooth muscle. Inaddition, there is a reduced amount of sarcospan, α-, δ-, γ-, andε-sarcoglycan in the sarcolemma of skeletal and cardiac muscle of themouse. The reduction in the amount of the aforementioned molecules aredetermined by comparison to the amounts of these molecules in thecorresponding tissues of a mouse lacking a disrupted β-sarcoglycan gene.

[0018] One of skill in the art will recognize that functional disruptionof the β-sarcoglycan gene can be achieved by several approaches.Generally, a specific disruption of a β-sarcoglycan gene is made in aprogenitor (embryonic stem) cell of a mouse, a heterozygous mouse isproduced, and homozygotes are generated by mating the heterozygotes. Inone embodiment, the disruption in the is produced from a disruptionresults in a deletion of a region of about 7.5 kb, the region including1606 bp of intron 2, the entirety of exons 3, 4, 5, 6, introns 3, 4, and5, and also 498 bp immediately downstream of exon 6. This deleted regionof the mouse genome is replaced with a PGK-neomycin cassette as a markerfor neomycin resistance. Such a disruption can be produced byintroduction, into embryonic stem cells, of a DNA construct which has1800 bp of intron 2 and 6500 base pairs of sequences which begin 498base pairs directly downstream of exon 6 of the wild type β-sarcoglycangene, having a neomycin resistance gene inserted between these sequencesat a position where the intervening sequences which are replaced arelocated in the wild type mouse genome.

[0019] Preliminary examination of the Sgcd-null and Sgcb-null micereveals strikingly similar phenotypes. Both the Sgcd-null mice and theSgcb-null mice develop a severe cardiomyopathy with focal areas ofmyocardial ischemic like-lesions as the characteristic histopathologicalfeature, followed by fibrotic calcification and scarring of the cardiacmuscle. This is in contrast to the previously generated Sgca-null mouse(Duclos et al., J. Cell Biol. 142: 1461-1471 (1998)) which exhibits nohistopathological signs of cardiomyopathy, although the SG-SSPN complexwas absent in the cardiac muscle membranes of this mouse. These findingsare in accordance with the clinical data observed in patients withprimary mutations in the α-sarcoglycan gene, which also do not or onlymildly display cardiomyopathic phenotypes.

[0020] The most noticeable molecular difference between the Sgca-nullmouse and the Sgcd-null mouse and Sgcb-null mouse of the presentinvention is expression of the SG-SSPN complex in smooth muscle cells ofthe vasculature. While the expression of the SG-SSPN complex is reducedat the sarcolemma of skeletal and cardiac muscle in all three mutants,compared to expression of these molecules in a wild type mouse, theexpression of the sarcoglycan-sarcospan complex in smooth muscle cellsof the blood vessels is disrupted in the Sgcd-null and the Sgcb-nullmice, but not in the Sgca-null mice. These findings, together withobserved characteristic ischemic-like lesions in cardiac and skeletalmuscle of Sgcd-null and Sgcb-null mice, indicate that vasculardysfunction has an essential impact on the development of cardiomyopathyand on the severity of muscular dystrophy in these mutant mice. Theseobservations indicate the existence of a novel mechanism in thepathogenesis of cardiomyopathy where disruption of thesarcoglycan-sarcospan complex in vascular smooth muscle perturbsvascular function, induces ischemic injury in cardiac and skeletalmuscles, and leads to cardiomyopathy and muscular dystrophy exacerbationin human patients. The disclosed findings open up a new area of researchinto the functional role of the sarcoglycan-sarcospan complex invascular smooth muscle and its involvement in coronary artery disease.

[0021] Applicants have previously demonstrated gene replacement therapyfor the treatment of mammalian sarcoglycan deficient limb-girdlemuscular dystrophy in Campbell et al., U.S. Patent Application Ser. No.09/164,664, filed Oct. 1, 1998, currently pending, the contents of whichare incorporated herein by reference. This previous disclosuredemonstrated that sarcoglycan gene replacement therapy of a deficientsarcoglycan species into skeletal muscle cells of a patient producesextensive long-term expression of the deficient sarcoglycan species torestore the entire sarcoglycan complex, results in the stableassociation of α-dystroglycan with the sarcolemma, and eliminates themorphological markers of limb-girdle muscular dystrophy. The presentinvention indicates that additional therapeutic benefit can be producedin individuals having a deficiency of certain specific sarcoglycanspecies (e.g. δ-sarcoglycan, β-sarcoglycan, and possibly ε-sarcoglycan)from additionally directing gene replacement therapy towards smoothmuscle cells, especially vascular smooth muscle cells, of theindividual. Specifically, an individual suffering from a non-dominantdeficiency of δ-sarcoglycan (e.g. mammalian autosomal recessivelimb-girdle muscular dystrophy type 2F) is treated by introducing anexpression vector encoding a wild-type form of δ-sarcoglycan intoskeletal and smooth muscle tissue of the individual under conditionsappropriate for expression of the gene in the different tissues. Thismethod may optimally utilize two or more different expression vectors,to achieve the desired patterns of delivery and expression of theintroduced δ-sarcoglycan gene. Expression vectors currently known in theart are include, without limitation, adenovirus based expressionvectors, described by Gregory et al., (1997) U.S. Pat. No. 5,670,488;McClelland et al., (1998) U.S. Pat. No. 5,756,086; Armentano et al.,(1998) U.S. Pat. No. 5,707,618; Saito et al., (1998) U.S. Pat. No.5,731,172, the contents of each are incorporated herein by reference.Also included are gutted adenovirus delivery systems (Clemens et al.,Gene Therapy 3: 965-972 (1996)), and adeno-associated virus (AAV) basedvectors, some examples of which are described by Podsakof et al., (1999)U.S. Pat. No. 5,858,351; Carter et al., (1989) U.S. Pat. No. 4,797,368;Lebkowski et al., (1992) U.S. Pat. No. 5,153,414; Srivastava et al.,(1993) U.S. Pat. No. 5,252,479; Lebkowski et al., (1994) U.S. Pat. No.5,354,678; Wilson et al., (1998) U.S. Pat. No. 5,756,283, the contentsof each being incorporated herein by reference. Other possible geneexpression systems for sarcoglycan gene replacement therapy includeretroviral based vectors and delivery systems (Miller et al., Blood 76,271 (1990), Booth et al., (1995) U.S. Pat. No. 5,466,676, the contentsof which are incorporated herein by reference, and also plasmid basednucleic acid delivery systems described by Eastman et al., (1998) U.S.Pat. No. 5,763,270, the contents of which are incorporated herein byreference.

[0022] The method of delivery of the gene expression system to thetarget tissue should result in direct contact of the gene expressionsystem to the target tissue, and will vary with the expression systemused and the target tissue. Common methods of delivery are intramuscularinjection (e.g. for delivery to skeletal muscle), and intravenousadministration (e.g. for delivery to vascular smooth muscle cells).Administration of the deficient sarcoglycan gene should optimally occurat as early a stage in disease progression as diagnosis permits,preferably, prior to the onset of severe muscle or cardiovasculardamage. Genetic diagnosis of the disease prior to the onset of thepathology allows gene therapy intervention at an extremely early stagein life.

[0023] Preferably, tissue specific regulatory elements or promoterelements are utilized in the expression vector(s). Optimally, theregulatory elements are specific for expression in muscle, and mayfurther be specific for skeletal muscle or smooth muscle. Examples ofsuch tissue specific regulatory elements and methods of use in genetherapy are described in Ordahl et al., (1993) U.S. Pat. No. 5,266,488,and Olson et al., (1998) U.S. Pat. No. 5,837,534, the contents of eachbeing incorporated herein by reference.

[0024] An individual suffering from a non-dominant deficiency ofβ-sarcoglycan (e.g. mammalian autosomal recessive limb-girdle musculardystrophy type 2E) is likewise treated by introducing an expressionvector encoding a wild-type form of β-sarcoglycan into skeletal andsmooth muscle tissue of the individual, by the above described methods.

[0025] The development of the Scgd-null and Scgb-null mice providesanimal models for the human conditions of autosomal recessivelimb-girdle muscular dystrophy type 2F and 2E, respectively, and alsofor ischemic heart disease which is associated with a reduction inexpression of the sarcoglycan-sarcospan complex in the vascular smoothmuscle cells of an individual. These animal models more closely parallelthe human disease condition than animal models previously known.Recently, Hack and colleagues (Hack et al., J. Cell Biol. 142: 1279-1287(1998)) reported that mice deficient in γ-sarcoglycan developcardiomyopathy. The authors described primarily fibrotic changes in theventricular wall. However, there was no report of initial acute necroticareas in cardiac muscle and the authors suggested that thecardiomyopathy might be secondary to dystrophic changes of thediaphragm. Another animal model, the BIO 14.6 cardiomyopathic hamster,which has been shown to have a genomic deletion in the δ-sarcoglycangene (Sakamoto et al., FEBS Letters 447: 124-128 (1999)), displayscardiac abnormalities similar to the Sgcd-null mice and associated withmicrovascular dysfunction (Factor et al., Circulation 66: 342-354(1982)). However, the skeletal muscle of the cardiomyopathic hamster isnot as severely dystrophic as in the Sgcd-null mice. Recent geneticstudies revealed expression of δ-sarcoglycan transcripts in some tissuesof the BIO 14.6 hamster (Sakamoto et al., FEBS Letters 447: 124-128(1999)), indicating that the BIO 14.6 hamster may not be completelydeficient in the δ-sarcoglycan protein. These observations indicate thatthe differences in severity of the clinical phenotypes are due to thedifference in the type of the genetic lesion in each case.

[0026] These new animal models are useful in the identification oftherapeutic compounds for the treatment of similar human conditions.Therefore, another aspect of the present invention is a method foridentifying a therapeutic compound useful for treatment of an individualdiagnosed with a deficiency in wild type β-sarcoglycan or δ-sarcoglycan.The deficiency may result from a efficiency in gene expression, or adeficiency in protein function. Such deficiencies include, withoutlimitation, autosomal recessive limb-girdle muscular dystrophies of type2F and type 2E. To identify therapeutic compounds for the treatment of apatient with a β-sarcoglycan deficiency, a mouse which is homozygous fora disrupted β-sarcoglycan gene is used. A candidate compound isadministered to the mouse, and assays are performed to detect anytherapeutic effects on the mouse which result from administration of thecandidate compound. Therapeutic effects include, without limitation areduction or reversal in disease progression, compared to theappropriate controls, and/or an alleviation of disease symptoms,compared to the appropriate controls. A candidate compound whichproduces such therapeutic effects upon administration to the mouse is tobe considered a therapeutic compound for the treatment of an individual,usually a human, diagnosed with a β-sarcoglycan deficiency. To identifytherapeutic compounds for the treatment of δ-sarcoglycan deficiency, amouse which is homozygous for a disrupted δ-sarcoglycan gene is used inthis method.

[0027] This method is useful for screening a variety of candidatecompounds. Without limitation, candidate compounds include previouslyknown drugs, small molecules (e.g. from a library), or genes for use ingene therapy. A candidate compound may also be any combination of theseagents.

[0028] The method of administration of the candidate compound to themouse will depend upon the properties of the candidate compound and anyspecific therapeutic effects which may be desired. For instance, becausethe lack of either δ-sarcoglycan or β-sarcoglycan expression in smoothmuscle cells, especially vascular smooth muscle cells, contributes tocardiomyopathy, therapeutic effects regarding vascular disfunction areexpected to be gained by administration of a therapeutic compound bymeans to contact the compound with smooth muscle cells, especiallyvascular smooth muscle cells, of the mouse. In addition, administrationby means to contact a therapeutic compound with skeletal muscle cells,or cardiac muscle cells, of the mouse is also expected to havetherapeutic effects, since the genetic deficiency directly effects thesetissues as well. Such administration may be achieved, without limitationby either intravenous, intraperitoneal, intramuscular, oral, or topicaladministration. The determination of the appropriate mode ofadministration of a candidate compound is within the ability of one ofskill in the art through no more than routine experimentation.

[0029] Under circumstances where the candidate compound is a gene, thegene is to be administered under conditions appropriate for theexpression of the gene in cells of the mouse. One such way it toincorporate the gene into a mammalian expression vector and deliver theexpression vectors into target cells (e.g. smooth muscle cells, skeletalmuscle cells, cardiac muscle cells) of the mouse. Some expressionvectors promote integration of the gene into the genome of the targetcells, other expression vectors remain separate from the cellulargenome. Several potential expression vectors for use in this method aredescribed above.

[0030] Cells of the Sgcd- or Sgcb-null mice may be obtained from themice and propagated in culture for use in the identification oftherapeutic compounds for the treatment of an individual with arespective non-dominant δ-sarcoglycan or β-sarcoglycan deficiency,respectively. Particularly useful cells to use in such a method aresmooth muscle cells, especially vascular smooth muscle cells, and alsoskeletal muscle cells of the mouse. In the method, a candidatetherapeutic compound is administered to the cells and then the cells areassayed for therapeutic effects which result from this administration.Determination of therapeutic effects depends upon the pathology of thespecific cells used. For example, a partial or complete restoration ofthe dystroglycan complex may be used to indicate therapeutic effects inskeletal or cardiac muscle cells. Along the same lines, partial orcomplete restoration of the sarcoglycan-sarcospan complex, or anincrease in the amount of α-, ε-sarcoglycan, sarcospan, and/orα-dystroglycan compared to control cells may be used to indicatetherapeutic effects on smooth muscle cells.

[0031] Another aspect of the present invention relates to the treatmentof ischemic heart disease which is caused by, or associated with,reduced expression of the sarcoglycan-sarcospan complex in vascularsmooth muscle cells of an individual. Such a reduction in expression maybe due to a defect in one or both δ-sarcoglycan genes of the individual,or to a defect in one or both β-sarcoglycan genes of the individual. Oneof skill in the art will recognize that other defects can also producethis phenotype in an individual. The method comprises contacting thevascular smooth muscle cells of the individual with a vascular smoothmuscle relaxant. Experiments detailed in the Exemplification sectionbelow show that administration of the vascular smooth muscle relaxant,Nicorandil to Sgcd-null mice, prior to the application of physicalstress, prevented mortality and the development of multiple myocardiallesions in treadmill stressed Sgcd-null mice. This strongly indicatesthat administration of a vascular smooth muscle relaxant (e.g.Nicorandil, Verapamil, Nitroglycerine, Dipyridmole) to an individualwith reduced expression of the sarcoglycan-sarcospan complex in vascularsmooth muscle cells will produce similar therapeutic results.Substantial therapeutic benefit can be obtained when administrationprecedes the onset of physical stress of the individual. Acceptablemodes of administration include, without limitation, intraperitoneal,intravenous, subcutaneous, and oral administration. According topharmacological studies, dilation of coronary artery microvesselsdepends on potassium channel activation and is mainly observed at lowconcentrations of Nicorandil (Kaski, J. C., Cardiovasc. Drugs Ther. 9:221-227 (1992)). Therefore, it is preferable that the smooth musclerelaxants are administered at dose which ensures predominant action onthe coronary artery microvasculature, without significantly loweringblood pressure. The appropriate dosage and route of administrationvaries with the specific condition of each individual, and can bedetermined by the skilled practitioner through no more than routineexperimentation.

[0032] Administration of a vascular smooth muscle relaxant, by themethods described above may also be used to prevent ischemic injury inskeletal and cardiac muscle of an individual which has reducedexpression of the sarcoglycan-sarcospan complex in his vascular smoothmuscle cells. This type of ischemic injury results from conditions suchas mammalian autosomal recessive limb-girdle muscular dystrophy type 2Eor 2F. This method is also expected to be of therapeutic benefit to anindividual suffering from other such conditions which result in asimilar reduction of expression of the sarcoglycan-sarcospan complex incells of the individual. Increased benefit may be obtained whenadministration occurs prior to the experience of physical stress.

[0033] Another aspect of the present invention is a method foridentifying a therapeutic compound for the treatment of ischemic heartdisease in an individual caused by, or associated with, reducedexpression of the sarcoglycan-sarcospan complex in the vascular smoothmuscle cells of the individual. In the method, a candidate compound isadministered to a mouse which has reduced expression of thesarcoglycan-sarcospan complex in the vascular smooth muscle cells (e.g.a Sgcd-null or Sgcb-null mouse). Administration is by means to contactthe candidate compound with the vascular smooth muscle cells of themouse, similar to that in the above described methods. The mouse is thenassayed for therapeutic effects which arise in response toadministration of the candidate compound. Therapeutic effects include,without limitation, a reduction or reversal in the accumulation ofischemic injury, and or a reduction of symptoms which arise fromischemic injury. Therapeutic effects are detected by comparison of themouse condition to the condition of the appropriate control mice. Adetection of therapeutic effects is an indication that the administeredcompound has therapeutic properties.

[0034] Another aspect of the present invention is a method foridentifying a therapeutic compound for the prevention of ischemic injuryin skeletal and cardiac muscle of an individual, wherein the injury iscaused by or associated with reduced expression of thesarcoglycan-sarcospan complex in vascular smooth muscle cells of theindividual. Similar to the above described methods, a mouse which hasreduced expression of the sarcoglycan-sarcospan complex in its vascularsmooth muscle cells (e.g. a Scgd-null or Scgb-null mouse) is used toscreen candidate compounds. A candidate compounds is administered to themouse by means to contact the vascular smooth muscle cells of the mouse,as describe above. The mouse is then assayed for a decrease in theischemic injury in skeletal and cardiac muscle which accumulates,attributable to the administration of the compound, by comparison to theappropriate control mice. It may be of benefit to subject the mouse tostress (e.g. using treadmill exercise) to accelerate ischemic injury, tomore rapidly detect injury prevention. The detection of therapeuticeffects are an indication that the administered compound has protectiveproperties. Administration is accomplished by the above describedmethods.

[0035] The molecular mechanism for the vascular dysfunctions observed inSgcd-null mice and Sgcb-null mice remains to be determined. Recentbiochemical evidence suggests the presence of at least threeinterconnected subcomplexes, dystrophin, dystroglycan and sarcoglycanwithin the DGC (Crosbie et al., J. Cell Biol. 145: 153-165 (1999)). Theresults indicate that the expression of each of these subcomplexes is aprerequisite for the functional structural membrane association of theDGC. It may be possible that the absence of the SG-SSPN complex leads tostructural and/or conformational changes of the remaining components ofthe DGC, which may be related to the abnormal contraction and/ordilation of the vascular smooth muscle. Yet another possibility is thatmetabolic and signalling pathways are involved in the microvasculardysfunction. Elevated levels of intracellular calcium, disturbances ofthe nitric oxide synthase pathway as well as increased activity ofprotein kinase C have been implicated in increased contractility and/orspasm of the microvasculature.

Exemplification Section I

[0036] DISRUPTION OF THE δ-SARCOGLYCAN GENE LEADS TO DISRUPTION OF THESARCOGLYCAN-SARCOSPAN COMPLEX IN VASCULAR SMOOTH MUSCLE AND REVEALS ANOVEL MECHANISM IN THE PATHOGENESIS OF CARDIOMYOPATHY AND MUSCULARDYSTROPHY

Generation of the Sgcd-null Mice

[0037] In order to create Sgcd-null mice, a targeting vector wasdesigned to replace exon 2, which encodes 63 amino acids of theintracellular domain and the entire transmembrane domain (FIG. 1).Southern blot analysis was performed on 370 neomycin resistant EScolonies which had received the targeting vector to identify cloneswhich had appropriately integrated the targeting vector DNA. DNA fromthe ES colonies was digested with EcoRI or BamHI/XhoI and probed withprobe A (FIG. 1) and B (FIG. 1), respectively. Probe A was seen tohybridize with a new 8.8-kb fragment, which was generated by the correctreplacement of exon 2 by the neo cassette, in addition to the 7-kb wildtype fragment. Probe B was seen to hybridize with a 5-kb fragment whichwas produced by digestion of the XhoI site introduced by the correctplacement of the neo cassette, in addition to the 7.5-kb wild typeallele. This analysis revealed homologous recombination in 7 independentclones. Two of these heterozygous clones were then used to producechimeric founder mice. Heterozygous mice from the F1 generation werecrossed to obtain Sgcd-null mice and the offspring were tested for exon2 deletion by Southern blot and PCR analysis. PCR analysis of tail DNApurified from wild type, heterozygous, and Sgcd-null mice produced a 600bp band corresponding to the wild type allele, and a 700 bp bandcorresponding to the null allele. The number of homozygous mutantoffspring obtained was the expected 25%, based on Mendelian inheritance.Northern blot analysis, using the complete cDNA coding sequence ofδ-sarcoglycan gene as a probe, revealed a transcript of 9-kb in theskeletal muscle of wild type, heterozygous, and homozygous Sgcd-nullmice. An additional hybridization with a probe specific for exon 2identified the 9-kb transcript only in the wild type and heterozygous,but not in the mutant mice, indicating a deletion of exon 2 from bothalleles of the mutant mice. RT-PCR analysis performed with a forwardprimer in exon 1 and a reverse primer in exon 5, revealed a PCR productrepresenting the normal transcript (600 bp) in wild type andheterozygous mice and an additional PCR product (400 bp) in heterozygousand mutant mice. Sequencing of this PCR product suggested thatalternative splicing occurred between exon 1 and exon 3 of theδ-sarcoglycan gene. In this case, an open reading frame from exon 3 toexon 8 would be maintained. Translation of this smaller transcript wouldproduce a 218 aa protein, lacking the entire transmembrane domain andpart of the N-terminus. However, no protein was detected in the skeletaland cardiac muscle fibers of the Sgcd-null mice by western blot of totalhomogenates and KCl washed microsomes by using an affinity purifiedpolyclonal antibody directed against the C-terminal or N-terminalportion of δ-sarcoglycan. Immunohistochemical analysis of skeletal andcardiac muscle revealed a complete absence of δ-sarcoglycan with theconcomitant loss of the SG-SSPN complex. Overall, the Sgcd-null micewere fertile and females able to bear at least two litters. Preliminarydata indicated an increased number spontaneous deaths in Sgcd-null mousecolony at around 6 months of age. Two founder mice were produced andanalyzed, both of which exhibited the same phenotypes.

Sgcd-null Mice Exhibit a Severe Muscular Dystrophy

[0038] In order to examine the effect of targeted disruption of theδ-sarcoglycan gene on skeletal muscle morphology, hematoxylin and eosin(H&E)-stained sections of the calf, thigh, and diaphragm muscles wereevaluated in wild type (n=8), Sgca-, Sgcd-heterozygous (n=26) andSgcd-null mice (n=26) between the ages of 2 weeks and 6 months.Sgcd-heterozygous mice did not show any morphological abnormalities.Interestingly, the skeletal muscle of the Sgcd-null mice, even in thevery young animals (n=8), showed extensive pathological alterations. Thepredominant feature was consistent with various stages of skeletalmuscle necrosis or regeneration similar to pathological alterationsobserved in tissue infarcts. Severe dystrophic changes were evident inskeletal muscle at all three sites from a very early age (1 month).Areas of tissue regeneration had a large percentage of myocytes withcentrally placed nuclei. Regions of full thickness necrosis orregeneration were seen in the diaphragms of these mice. Chronicdystrophic changes accumulated with age in all of these same musclegroups. Large regions of necrosis/regeneration were observed in calf andthigh muscles of mice at all ages. Severe necrotic lesions includingcentral nucleation, endomysial fibrosis, atrophy, hypertrophy, and fattyinfiltration were predominantly seen in the diaphragm in younger animalsat 1 month of age. Similar findings were observed in a second founderfrom another cell line of targeted ES cells. Based on the evaluation of200-1,100 myofibers per muscle, 80-100% of nonregenerating myocytescontained internally placed nuclei by the age of 1 month (n=4). Inaddition to the described severe necrotic/regenerative lesions, a broadspectrum of other dystrophic changes were observed in older (>4 months)Sgcd-deficient muscle (n=18). These changes included endomysialfibrosis, fiber splitting, hypertrophy, dystrophic calcification andfatty infiltration. Evaluation of creatine kinase (CK) levels inSgcd-null mice revealed a 15-20-fold elevation of CK as compared to wildtype mice.

Sgcd-null Mice Display Severe Cardiomyopathy

[0039] In order to evaluate whether disruption of the δ-sarcoglycan genemay cause cardiac abnormalities, H&E staining of transverse sections ofhearts from wild type (n=8) and Sgcd-null mice between the ages of 2weeks and 6 months (n=26) was performed. From 2 weeks to 3 months of age(n=8), hearts from Sgcd-null mice were nearly normal and only rare,small foci of necrosis were seen. Myocardial tissue studied after 3months of age revealed more extensive alterations (n=18). Larger andmore numerous foci of active cellular necrosis and granular calciumdeposits involving small groups of myocytes were present. These fociwere sharply demarcated from surrounding tissue, which appeared to benormal. The localization and extent of pathology predilection sitesvaried considerably from animal to animal. In some hearts subendocardialregions were predominantly affected, whereas in others, pathologicalchanges in the outer two-thirds of the free walls of both ventricleswere observed. In older animals (5-6 months) [n=14] active myocardialnecrosis was less evident, but various stages of calcification andfibrosis were observed. Interestingly, female mice that had beenpregnant at least once (n=4) displayed more widespread and advancedcardiac alterations than age-matched virgin females (n=4). In contrast,extensive histopathological evaluations of the myocardium of Sgca-nullmice (including pregnant females) showed no pathological alterationsbesides minimal calcification in some of the older animals. No coronaryvessel histopathology at the light microscopy level was observed.

Disruption of the SG-SSPN Complex in the Smooth Muscle of the CoronaryArteries in Sgcd-null Mice

[0040] The characteristic histopathological abnormalities suggested thatalterations in vascular smooth muscle might be responsible for thesefindings. Recent biochemical studies (Straub et al., J. Biol. Chem., inpress (1999)) suggest that the composition of SG-SSPN complex in smoothmuscle is distinct from that in skeletal and cardiac muscle.Consequently, immunohistochemical analysis was performed on componentsof the DGC in cardiac muscle fibers and smooth muscle cells of coronaryarteries from wild type, Sgca- and Sgcd-null mice. In wild type mice,α-, β-, γ-, δ-, and ε-sarcoglycan, sarcospan and β-dystroglycan werehomogeneously expressed at the cardiac muscle fiber membranes. β-, δ-and ε-sarcoglycan, sarcospan and β-dystroglycan were also stronglyexpressed in the smooth muscle cells of the coronary arteries. Incontrast, α- and γ-sarcoglycan were not expressed in smooth muscle cellsof coronary arteries. In cardiac muscle fibers of Sgca-null mice,α-sarcoglycan was absent from the sarcolemma, whereas δ-sarcoglycan wasabsent from the sarcolemma of Sgcd-null mice respectively. In addition,there was a concomitant loss of α-, β-, γ- and δ-sarcoglycan. Sarcospanwas absent from the sarcolemma of both animal models. Interestingly,ε-sarcoglycan was absent from the sarcolemma of Sgcd-null mice incontrast to wild type and Sgca-null mice. However, the most remarkabledifference between the Sgca- and the Sgcd-null mice was observed in theexpression of the SG-SSPN complex in the smooth muscle of coronaryarteries. While β-, δ-, and ε-sarcoglycan along with sarcospan werestill strongly expressed in the coronary arteries of the Sgca-null mice,these proteins were completely absent in the smooth muscle cells ofSgcd-null mice. The same expression pattern was observed in smoothmuscle cells of other blood vessels (e.g. the femoral artery). Stainingwith an antibody against smooth muscle actin confirmed the presence ofsmooth muscle in the vasculature. Western blot analysis of aortic tissueconfirmed the immunohistochemical observation that the SG-SSPN complexis disrupted in Sgcd-null mice, while it is still preserved in aortafrom wild type and Sgca-null mice. All analyses of knockout mice werecompared to wild type controls. Taken together, these results indicatethat targeted ablation of the δ-sarcoglycan gene leads to disruption ofthe SG-SSPN complex in smooth muscle, whereas the complex is preservedin smooth muscle cells of Sgca-null mice.

Coronary Artery Vascular Irregularities in Sgcd-null Mice

[0041] The Microfil® perfusion technique was used in vivo in order todetermine whether disruption of the SG-SSPN complex in smooth muscle ofcoronary arteries indeed leads to vascular perfusion abnormalities. Wildtype, Sgca-null and Sgcd-null mice at the age of 2-6 months wereperfused, and cleared sections of the heart were visualized usingtrans-illumination with low-power magnification. The coronarymicrovessels were distributed normally and were smoothly tapered in bothwild type and Sgca-null mice. Some animals showed areas of focal vesselnarrowing but never showed any severe irregularities. In contrast,Sgcd-null mice displayed numerous areas of pronounced constrictions.Pre- and poststenotic dilation as an appearance of microaneurysm wasfrequently associated with these constrictions. Extensive areas of focalvascular lumen narrowing and a generalized sparseness of perfusion wereobserved. Interestingly, although general perfusion was diminished incapillaries of Sgcd-null mice, no constrictions were observed.Quantification of vascular abnormalities in Sgcd-null mice at differentages were determined by calculating the mean numbers of abnormal vessels±SEM in 10 nonadjacent microscopic fields at a magnification of 10×. Nolesions were detected in Sgca-null and wild type mice. Analysis ofSgcd-null mice revealed irregularities in 2 (5±1.1), 4 (11±1.5) and 6months old mice (4±0.9) [n=10, in each age group respectively]. However,the most severe and abundant abnormalities were observed at the age of 4months, a time when acute necrosis was first observed in Sgcd-null mice.These results indicate that the disturbance of the vasculature precedesthe onset of myocardial ischemic lesions.

[0042] The Microfil® in vivo perfusion enabled the study of longsegments of coronary artery branches in three dimensions. Quantificationof perfusion abnormalities in Sgcd-null mice revealed that vesselirregularities were present even in young mice at a stage without anyovert signs of cardiac muscle necrosis. Moreover, the most severe andabundant perfusion abnormalities were observed at the time of acuteongoing necrosis, indicating that a certain degree of vasculardysfunction may be required to reach an ischemic threshold necessary toinduce myocardial necrosis. These data indicate that the lesionsobserved in Sgcd-null mice are not an induced epiphenomenon caused byalterations of the cardiac muscle per se. Rather, the data indicate thatincreased vascular tone comprises blood supply in a diffuse mannerleading to focal ischemic injury, necrosis and fibrotic changes.

Treadmill Exercise Initiates the Development of Cardiac Muscle Necrosisin Young Sgcd-null Mice

[0043] In order to test the hypothesis that the observed abnormalitiesof the vasculature represent a dynamic hyper-reactivity of thevasculature, which may be triggered by stress, wild type (n=20), Sgca-(n=20) and Sgcd-null mice (n=42) were exercised for 40 min using atreadmill. Treadmill exercise is one of the primary methods usedclinically to induce cardiovascular stress in human and animals (Fewellet al., Am. J. Physiol. 273: H1595-H1605 (1997)) and is used to detectcardiovascular abnormalities (e.g. coronary artery dysfunction) that maynot be readily apparent at rest. The exercised mice were studied at theage of 2-3 months a time where Sgcd-null mice do not show any overtsigns of cardiac muscle necrosis, but do have microvessel abnormalitiesas revealed by perfusion studies. All mice were injected with Evans bluedye (EBD) 8 hr before the exercise. EBD is a small molecular mass tracerthat tightly complexes with serum albumin. Normally, this is a membraneimpermeable molecule, but if the sarcolemma integrity is compromisedthis dye readily penetrates into the cytoplasm of muscle fibers (Matsudaet al., J. Biochem. (Tokyo) 118: 959-964 (1995); Straub et al., J. CellBiol. 139: 375-385 (1997)). EBD uptake as well as routine histopathologywas examined in the cardiac muscle. Interestingly, approximately ⅓ ofthe Sgcd-null mice died suddenly during the exercise while no deathoccurred in Sgca-null and wild type mice. The surviving Sgcd-null micewere sacrificed 36-48 hr after exercise for histological assessment ofcardiac muscle.

[0044] All mice displayed multiple areas of EBD uptake corresponding toacute histopathological features of necrosis as revealed by H&Estaining. Histological analysis of these myocardial lesions displayedcoagulation necrosis, which is a characteristic histological featureobserved in conditions associated with myocardial ischemia. No signs ofEBD uptake from necrosis were detected in age matched non-exercisedSgcd-null or wild type mice. Only a few single necrotic cells wereobserved in Sgca-null mice. Quantification of Evans blue staining aftertreadmill exercise revealed 13-27% positive stained areas in cardiacmuscle sections of Sgcd-null mice and less than 3% positive stainedareas in Sgca-null mice.

[0045] In order to demonstrate that these observations were related tovascular dysfunction, a vascular smooth muscle relaxant compound,Nicorandil, was administered to the Sgcd-null mice. Nicorandil has beenshown to relax coronary vascular smooth muscle by activation ofpotassium channels resulting in hyperpolarization of the smooth musclemembrane as well as by increasing cyclic GMP levels (Kukovetz et al., J.Cardiovasc. Pharmacol. 20 (Suppl.3): S1-S7 (1992)). In addition,Nicorandil has been shown to prevent coronary artery vasospasms under avariety of conditions (Kaski, J. C., Cardiovasc. Drugs Ther. 9: 221-227(1992)).

[0046] Intraperitoneal administration of Nicorandil, a vascular smoothmuscle relaxant, was able to prevent the development of multiplemyocardial ischemic lesions in all Sgcd-null mice (n=20) studied. No EBDuptake was observed in cardiac muscle of 2 months old exercisedSgcd-null mice after intraperitoneal application of Nicorandil for 3days prior to the exercise. Microfil® perfusion of coronary arteries inSgcd-null mice after administration of Nicorandil revealed no evidenceof vascular constrictions and displayed smoothly tapered branches of thecoronary vascular bed. In addition, there was overall greater density ofthe vasculature. Nicorandil, at the dose given, did not lower thesystemic blood pressure in Sgcd-null mice. No alteration of the generalbehavior during exercise or any cardiac muscle abnormalities wasobserved during or after exercise of wild type or Sgca-null mice afteradministration of Nicorandil. In addition, perfusion studies inNicorandil treated Sgcd-null mice showed the coronary microvascular bedfree of constrictions and focal luminal narrowing. The functionaldisturbance of the vasculature was demonstrated to initiate ischemicmyocardial necrosis. As the mice aged, this damage developed into asevere cardiomyopathy.

Methods of the Invention, Section I

[0047] Isolation of mouse δ-SG genomic and cDNA clones. One RT-PCRproduct from mouse skeletal muscle RNA was obtained using the hamsterprimer HadFor5 (5′-AGCTCAGAGGGGCCACAC-3′ (SEQ ID NO: 1), exon 2) and themouse primer MdRev2 (5′ -CAGCCAGTGTTTCAAGCCAA-3′ (SEQ ID NO: 2), exon8). This product, containing exons 2-8 of the δ-sarcoglycan gene, wasused to screen a Stratagene 129/SV mouse genomic library in vectorλFIXII (La Jolla, Calif.). Three positive clones were subcloned intopBlueScript KS (+) and restriction enzyme mapped using standardprocedures.

[0048] Generation of Sgcd-null Mice. The δ-SG targeting vector wasconstructed using the positive-negative selection vector pPNT. EcoRI andBamHI sites were introduced by high fidelity PCR mutagenesis (Takaraenzyme) at the ends of a 6-kb fragment that contains part of the δ-SGintron 1 and exon 1 b. The DNA was digested by EcoRI/BamHI and insertedin between the tk and neo genes of the vector. The second insert wasobtained by subcloning a 5-kb NotI-EcoRI δ-SG intron 2 fragment into thedigested NotI-EcoRI pBlueScript. The insert was isolated by NotI-XhoIdigestion and cloned into the NotI and XhoI sites of the plasmid. 2992bp were deleted from the genome of the recipient cell after homologousrecombination, and replaced by the neo gene. This deletion included 2277bp of intron 1, all of exon 2, and 576 bp of intron 2 of the δ-SG (FIG.1). The construct introduced into the recipient cells contained 5 kb ofintron 1 and 5 kb of intron 2, and lacked exon 2 of the δ-sarcoglycangene, which was replaced with a neomycin resistance gene insertedbetween the two introns in the opposite transcriptional orientation asthe δ-sarcoglycan exon which was replaced. R1 embryonic stem cells (ES)were grown and electroporated with 10 μg of the NotI linearizedtargeting plasmid. Colonies surviving G418 and Gancyclovir wereisolated, expanded, and screened by Southern blot analysis forappropriate incorporation of the vector DNA. ES cell lines from twodifferent, correctly targeted clones were injected into C57BL/6Jblastocysts and transferred into pseudopregnant females. After germ-linetransmission, DNA was extracted from the offspring's tails and thegenotyping was done by PCR using the following three different primersin the same reaction: NeoTR (5′-GCTATCAGGACATAGCGTTGGCTA-3′ (SEQ ID NO:3); Mdint1F (5′-GCAAACTTGGAGAGTGAAGAGGC-3′ (SEQ ID NO: 4); and Mdint1R(5′-GAGGCATATAAAGTTTGCACGAC-3′ (SEQ ID NO: 5)).

[0049] Northern Blot Analysis. Total RNA was isolated from wild type,δ-SG +/−, and δ-SG −/− skeletal muscle tissue using RNAzol B (Tel-Test)according to the manufacturer specifications. 20 μg of the RNA wassubjected to electrophoresis on a 1.25% agarose gel containing 5%formaldehyde, blotted to Hybond membrane (Amersham), and hybridized witheither a 760 bp exon 2-8 probe or an exon 2 probe from mouse δ-SG cDNA.

[0050] Histopathology studies. Wild type mice (n=8), Sgca- (n=26),Sgcd-heterozygous (n=26) and Sgcd-null mice (n=26) were anaesthetizedwith pentobarbital (0.75 mg/10 g of body weight) via intraperitonealinjection. Subsequently, the animals were perfused with PBS (15 ml)followed by 15 ml of 10% buffered formalin fixative solution. Afterembedding the tissue in paraffin, hematoxylin and eosin (H&E) stainedsections (4 μm) were prepared in order to characterize skeletal andcardiac muscle pathology. Some animals were sacrificed by cervicaldislocation and H&E staining was performed on cryosections of skeletaland cardiac muscle. Furthermore, H&E sections of brain, lung, liver,kidney and spleen were performed in some animals. No histopathology wasobserved in these non-muscle tissues. Creatine kinase values weredetermined in blood serum from wild type and Sgcd-null mice using thecreatine kinase assay kit from Sigma.

[0051] Immunofluorescence Analysis. Hearts and skeletal muscle wereisolated from wild type, Sgca- and Sgcd-null mice and rapidly frozen inliquid nitrogen cooled isopentane. 7 μm cryosections were prepared andanalyzed by immunofluorescence using different antibodies as describedpreviously (Duclos et al., J. Cell Biol. 142: 1461-1471 (1998)).

[0052] Antibodies. Rabbit polyclonal antibodies against α-sarcoglycan(rabbit 98), dystrophin (rabbit 31), the laminin α2 chain, β-sarcoglycan(goat 26), and δ-sarcoglycan N- and C-terminal peptide (rabbit 214 and229, respectively), α-dystroglycan fusion protein D (goat 20), andagainst β-dystroglycan C-terminal peptide (rabbit 83), sarcospan andε-sarcoglycan (rabbit 235 and 232, respectively) were describedpreviously (Duclos et al., J. Cell Biol 142: 1461-1471 (1998)). Anaffinity purified rabbit polyclonal antibody was produced against aCOOH-terminal fusion protein (aa 167-291) of γ-sarcoglycan. Acommercially available mouse monoclonal antibody was used to detectsmooth muscle actin (Sigma).

[0053] Microfil® perfusion. In order to study coronary microvascularperfusion mice were anesthetized with Phenobarbital 75 mg/kg body weightand a bilateral sternum incision was performed to expose the leftatrium. 1-1.5 ml of Microfil®, a liquid silicon rubber (Flow tech,Carver, Mass.) was perfused into the left atrium. The heart continuedbeating for about 1 min and after contraction stopped the heart wasrapidly excised and cured on ice for about 10 min. Adequacy of vascularperfusion was judged by the white blush that developed in the myocardiumas well as the white filling of other main arteries (mesenteric arteryand femoral artery) of the mouse body. The heart was fixed in 10%formalin for 24 hr and the next day the tissue was sliced into 2 mmthick transverse cross sections and cleared by sequential 24-himmersions in 25, 50, 75, 95 and finally 100% ethyl alcohol. On day 6,specimens were placed in pure methyl salicate for 12-24 hr.Microvascular perfusion was visualized with both epi- andtans-illumination and examined under low power magnification (×10-20).the vascular irregularities in Sgcd-null mice at different ages (2, 4,and 6 months) were quantified by counting the number of abnormalindividual vessel segments in 10 nonadjacent microscopic field using alow magnification (10×) and the mean number of abnormal vessels werecalculated for each mouse. An average number of abnormal vessels ±SEMwere then calculated for each age group. Vessels segments with more thanone abnormality were only counted once.

[0054] Treadmill exercise. Animals were exercised using the OmnipacerTreadmill Model LC4/M-MGA/AT, Accuscan Instruments, Inc., which had anadjustable belt speed (0-100 m/min), shock bars with adjustable amperageand an on-and-off shock switch for each lane. Animals were exercised at12-17 m/min for about 10 min and for 25-30 m/min for the remaining 50min. If an animal became exhausted, the shock bar of this lane wasturned off and the animal was allowed to rest at the back of thetreadmill for a short period of time. Wild type (n=20), Sgca- (n=20) andSgcd-null mice (n=42) were divided in approximately equal numbers ofmale and females. All mice were injected with Evans blue dye (0.5 mgEBD/0.05 ml PBS) intraperitoneally 8 hr before the exercise. Animalswere injected with 50 μl of this solution per 10-g body weight. Allsurviving animals were kept alive for 36-48 hr and serial sections ofcardiac muscle were studied for Evans blue uptake and forhistopathological signs of necrosis by using routine H&E technique. Theeffect of Nicorandil treatment on treadmill performance in wild type(n=6), Sgca- (n=6) and Sgcd-null mice (n=20) was studied after 3 days ofintraperitoneal injection of Nicorandil at a dose of 1 mg/kg body weighttwice a day. Quantification of Evans blue positive stained areas insections of cardiac muscle from Sgca- and Sgcd-null mice (n=20, eachgroup) was done by using the Scion image program. The percentage ofpositive stained areas was calculated by dividing the area of stainingby the total area of the analyzed heart section.

SECTION II

[0055] DISRUPTION OF THE β-SARCOGLYCAN GENE

Generation of Sgcb-null Mice

[0056] A P1-clone containing the mouse β-sarcoglycan gene wascharacterized in order to design a targeting vector for the generationof Sgcb-null mice. Murine and human β-sarcoglycan are highly homologousat the amino acid level and the structural organization of the gene intosix exons is shared by both species (GenBank/EMBL/DDBJ accession numberAF169288). Given that most human mutations have been found in exons 3,4, 5 and 6, which encode part of the transmembrane domain and theextracellular portion of β-sarcoglycan (Bönnemann et al., Nat. Genet.11: 266-273 (1995); Lim et al., Nat. Genet. 11: 257-265 (1995);Bönnemann et al., Hum. Mol. Gen. 5: 1953-1961 (1996); Bönnemann et al.,Neuromusc. Disord. 8: 193-197 (1998); Duclos et al., Neuromusc. Disord.8: 30-38 (1998)), exons 3, 4, 5 and 6 were targeted to create a mutantallele of Sgcb representative of human mutations. Homologousrecombination replaced exons 3 through 6 with the phosphoglyceratekinase promotor/neomycin phosphotransferase cDNA (FIG. 2). A total of361 colonies of ES cells surviving G418 and gancyclovir selection wereanalyzed by Southern blotting for the presence of homologousrecombination. DNA from the ES cells was prepared and digested with HindIII or Xba 1 and probed by Southern blot with probe 1 (see FIG. 2) and 2(see FIG. 2) respectively. The correct replacement of exons 3-6 by theneo cassette of the targeting construct produced a new 3.9 kb Hind IIIfragment, in addition to the 7.4 kb wild type Hind III fragment, whichwas identified with probe 1. correctly targeted clones were identified.Three of these clones were used to produce chimeras for germlinetransmission. Southern blot analysis of tail DNA from the heterozygousand homozygous progeny produced revealed the same bands as seen in theES cells, confirming the disruption of the β-sarcoglycan gene.Heterozygous mice appeared normal and homozygous mice were produced inexpected numbers in accordance with Mendelian inheritance. The effect ofthe mutation on β-sarcoglycan RNA was assessed by Northern blotanalysis. A cDNA probe specific for exon 2 (probe 1, FIG. 2) was used toprobe RNA extracted from skeletal muscle of wild-type, heterozygous, andSgcb-null mice. This probe detected the previously describedβ-sarcoglycan transcripts of 4.4, 3.0, and 1.4 kb in both wild-type andheterozygous mice. In contrast, none of the known transcripts weredetected in Sgcb-null mice. A faint transcript of 4 kb, however, wasdetected in Sgcb-null mice. This transcript may represent a transcriptcontaining exons 1 and 2 and the neo-cassette, however, attempts toamplify such a transcript with RT-PCR have been unsuccessful. A cDNAprobe specific for exon 6 (probe 2, FIG. 2) did not detect anyβ-sarcoglycan transcripts in the Sgcb-null animals, but did detect theexpected bands in wild-type and heterozygous animals.

[0057] Western blot and immunofluorescence analysis were also performedon progeny mice to analyze β-sarcoglycan protein expression. Monoclonaland polyclonal antibodies specific for the N-terminus of β-sarcoglycan(epitopes between amino acids 1-65, encoded from exon 1 and parts ofexon 2) were used to probe membrane-enriched preparations of skeletal,cardiac and lung membranes of wild-type, heterozygous, and Sgcb-nullmice, by Western blot analysis. The analysis detected β-sarcoglycanprotein in wild-type and heterozygous mice, but not in Sgcb-null mice.Immunofluorescence analysis detected β-sarcoglycan at the sarcolemma inwild-type skeletal and smooth muscle, but not at the sarcolemma ofskeletal or smooth muscle of Sgcb-null mice. Two founder mice wereproduced and analyzed, both of which exhibited the same phenotypes.

Sgcb-null Mice Develop a Severe Muscular Dystrophy and Cardiomyopathy

[0058] To evaluate the consequences of β-sarcoglycan deficiency,hematoxylin and eosin (H&E)-stained sections of the calf, thigh anddiaphragm muscle in wild type, heterozygous and Sgcb-null mice wereexamined. Histopathological features of muscular dystrophy were neverobserved in wild-type or heterozygous animals. In Sgcb-null mice,however, pronounced morphological changes were detected. Severedystrophic changes were observed in the Sgcb-null mice. Large areas ofnecrosis were observed in calf, thigh and diaphragm muscles of mice atall ages. Other dystrophic changes included internally placed nuclei ofnon-regenerating fibers (based on the evaluation of 200-1,100 myofibersper muscle, 80-100% of non-regenerating myocytes contained internallyplaced nuclei at the age of 2 months), fiber splitting and hypertrophy,extensive dystrophic calcification, endomysial fibrosis and massivefatty infiltration. Sgca-null mice have previously been generated(Duclos et al., J. Cell Biol. 142: 1461-1471 (1998)) and it isinteresting to note that upon comparison, the skeletal muscle pathologywas much more severe in Sgcb-null mice. For example, large areas ofnecrosis and fatty infiltration were not detected in Sgca-null mice.

[0059] Consistent with the severe dystrophic pattern, 13-16wk-oldSgcb-null mice displayed elevated serum creatine kinase activitycompared to age-matched wild-type and heterozygous mice. EBD injectionsinto 9-wk-old Sgcb-null mice revealed uptake of the blue dye in variousskeletal muscles, indicating compromised sarcolemma integrity, whereasno EBD staining was seen in control mice.

[0060] Dystrophin defects, including Duchenne or Becker musculardystrophies, are also manifested at the cardiac level (Towbin, J. A.,Curr. Opin. Cell Biol. 10: 131-139 (1998)). Less is known about theheart involvement in muscular dystrophies caused by sarcoglycanmutations. To evaluate the consequences of β-sarcoglycan deficiency inthe heart H&E stainings of transverse sections of hearts from wild-type,heterozygous and Sgcb-null mice was performed. No cardiac abnormalitieswere observed in control mice of any age. In sharp contrast, smallnecrotic areas already in 9-wk-old hearts from Sgcb-null mice weredetected. Similar histological analysis of hearts of 20-wk-old Sgcb-nullanimals revealed more extensive alterations. Prominent necrotic areas,resembling ischemic-like lesions, were present throughout the right andleft ventricle. In 30-wk-old animals, active myocardial necrosis wasless evident and instead widespread areas of fibrosis were detected.

β-Sarcoglycan-Deficiency Causes Loss of the Sarcoglycan-SarcospanComplex, the Dystroglycan Complex and ε-Sarcoglycan in Skeletal, Cardiacand Smooth Muscle

[0061] Immunofluorescence analysis for each component of the DGC wasperformed in order to analyze the consequences of a β-sarcoglycandeficiency in skeletal, cardiac and smooth muscle at the molecularlevel. Skeletal muscle cryosections from wild-type and Sgcb-null mice (4week old) were stained independently with antibodies againstα-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan,ε-sarcoglycan, sarcospan, dystrophin, α-dystroglycan, β-dystroglycan,and laminin α2 chain. Immunofluorescence analysis revealed thatβ-sarcoglycan was absent from the sarcolemma of skeletal muscle fibersin Sgcb-null mice. Also, α-, γ-, and δ-sarcoglycan were concomitantlyreduced along with sarcospan. Interestingly, ε-sarcoglycan was alsoreduced at the sarcolemma. Dystrophin staining appeared normal inSgcb-null mice. α- and β-dystroglycan appeared slightly reduced whereasthe laminin α2 chain was present at comparable levels with controlskeletal muscle. Similar results were also obtained in heart.

[0062] To evaluate if the absence of β-sarcoglycan also affected theexpression of the other sarcoglycans and sarcospan in smooth muscle,immunofluorescence analysis was performed on lung smooth muscle. Lungcryosections from wild-type and Sgcb-null mice (4 week old) were stainedindependently with antibodies against α-, β-, γ-, δ-, and ε-sarcoglycan,and also sarcospan. This analysis revealed that β-, δ- and ε-sarcoglycanand sarcospan were all expressed in lung smooth muscle of pulmonaryarteries in wild-type mice whereas α- and γ-sarcoglycan were notdetected. Absence of β-sarcoglycan in vascular smooth muscle alsoaffected the expression of the smooth muscle sarcoglycans along withsarcospan, which was concomitantly greatly reduced.

[0063] To further examine the expression of DGC components, immunoblotanalysis was performed on isolated membrane preparations from wild-type,heterozygous and homozygous mutant skeletal, cardiac and smooth muscle.Skeletal, cardiac and lung KCl-washed microsomes from wild-type,heterozygous and Sgcb-null mice were analyzed by 3-12% SDS-PAGE andimmunoblotted with antibodies against the sarcoglycans (α-, β-, γ-, δ-,and ε-), sarcospan, dystrophin, and α-dystroglycan. Blots were alsoprobed with antibodies against the α2 subunit of the dihydropyridinereceptor to verify equal loading of protein samples.

[0064] In accordance with the immunofluorescence analysis, β-sarcoglycanwas determined to be absent in skeletal and cardiac muscle of Sgcb-nullmice by Western blot analysis. Heterozygous mice expressed levels ofβ-sarcoglycan similar to the wild type control. Furthermore, α-, γ-, andδ-sarcoglycan were concomitantly reduced in both skeletal and cardiacmuscle from Sgcb-null mice. Lung tissue was used as a source for smoothmuscle. As expected, β-sarcoglycan was deficient in lung of Sgcb-nullmice. As noted above, α-sarcoglycan is not expressed in smooth muscle.

[0065] Although, as discussed above, γ-sarcoglycan was not detected inany cell-type in the lung by immunofluorescence, γ-sarcoglycan wasdetected by western blot analysis of wild-type and heterozygous lungtissue. This same analysis indicated that γ-sarcoglycan expression wasgreatly reduced in Sgcb-null lung tissue. In addition, δ-sarcoglycanlevels were significantly reduced in smooth muscle of Sgcb-null mice.Furthermore, ε-sarcoglycan and sarcospan levels were greatly reduced inthe three muscle lineages of Sgcb-null mice.

[0066] In agreement with the immunofluorescence analysis, α-dystroglycanlevels were determined to be greatly reduced in skeletal muscle ofSgcb-null mice, but Western blot analysis. In the supernatant fromSgcb-deficient skeletal muscle membrane preparations, α-dystroglycan wasenriched and fully glycosylated, but obviously failed to be stablyanchored to the membrane without the sarcoglycans. In cardiac muscle ofSgcb-null mice, α-dystroglycan was moderately reduced. Dystrophin wasmoderately reduced in skeletal muscle of Sgcb-null mice, not altered incardiac muscle, but greatly reduced in smooth muscle.

Vascular Irregularities in Sgcb-null Mice

[0067] The above observations indicate that a deficiency ofβ-sarcoglycan in vascular smooth muscle leads to a loss of thesarcoglycan-sarcospan complex in smooth muscle. The predominantcharacteristic feature of the muscular dystrophy and cardiomyopathy wasfocal areas of necrosis, resembling the pathological observations seenin tissue infarcts, occurring in ischemic injury. Therefore, loss of thesmooth muscle complex in the vasculature and the presence of necroticareas prompted investigation into whether the presence of abnormalitiesin the vasculature contributed to the pathological changes of skeletaland cardiac muscle. The Microfil® perfusion technique was used in vivoto study the organization of various vascular beds in skeletal andcardiac muscle of the mutant mice. Wild-type and Sgcb-null mice of 4weeks of age were perfused and cleared sections of the diaphragm andheart were analyzed using trans-illuminations with low-powermagnification. Interestingly, Sgcb-null mice exhibited numerous areas ofvascular constrictions often associated with pre- and poststenoticaneurysm in the vasculature of both diaphragm and heart, which was neverdetected in wild-type mice. In addition, the vessels of Sgcb-null miceexhibited a serrated contour rather than smoothly tapered vessel wallsthat are seen in wild-type mice. Functional disturbance of the coronaryartery microvasculature was detected at an age of 4 weeks, before anyovert signs of cardiac morphological alterations were observed.Similarly, vascular irregularities in the diaphragm were observed in4-wk-old Sgcb-null mice, at a time when acute necrosis starts to occurin the skeletal muscle. These observations indicate that the disturbanceof the vasculature precedes the onset of ischemic-like lesions inSgcb-null mice.

Presence of a Distinct E-Sarcoglycan Complex in Skeletal Muscle

[0068] Although ε-sarcoglycan is expressed in skeletal muscle (Ettingeret al., 1997; McNally et al., 1998) there are no reports ofε-sarcoglycan being associated with the skeletal muscle sarcoglycans. Inthe membrane preparations of Sgcb-null mice, ε-sarcoglycan was observedto be greatly reduced, suggesting that ε-sarcoglycan could be part of askeletal muscle sarcoglycan complex. To test this hypothesis DGC wasisolated from skeletal muscle of wild-type mice, Sgca-null and Sgcb-nullmice. The skeletal muscle DGC was extracted by digitonin and furtherpurified by WGA affinity chromatography followed by centrifugation ofthe skeletal muscle DGC through sucrose gradients. The migration of theDGC complex during high-speed centrifugation through sucrose gradientshas previously been demonstrated (Crosbie et al., FEBS Lett. 427:270-282 (1998)). Proteins from the sucrose gradient fractions wereseparated by SDS-PAGE using 3-12% polyacrylamide gels. Nitrocellulosetransfers of identical samples were probed with antibodies against theα-, β-, and γ-sarcoglycans, and α- and β-dystroglycan. α-, β, γ- andδ-sarcoglycan were observed to migrate in fractions 7-9 in wild-typemice. Western blotting of the same fractions with antibodies againstε-sarcoglycan demonstrated that ε-sarcoglycan co-migrated in the samefractions as the other sarcoglycans along with α- and β-dystroglycan,although a peak of α-dystroglycan was also seen in earlier fractions. Inthe Sgca-null mice (deficient in α-sarcoglycan) α-sarcoglycan wasabsent, and β-sarcoglycan was greatly reduced. Some γ- and δ-sarcoglycanremained but peaked in earlier fractions (5-7 instead of 7-9).ε-sarcoglycan, however, remained in fractions 7-9. In contrast,α-dystroglycan was absent in fractions 7-9, but was still present in theearlier fractions. β-dystroglycan was also absent from fractions 7-9,but some β-dystroglycan was still present in fractions 4-6, although theremaining β-dystroglycan was not associated with the remainingα-dystroglycan or the remaining γ-sarcoglycan. Together, these resultsindicate that deficiency of α-sarcoglycan causes dissociation of thesarcoglycan and dystroglycan complexes, without affecting the presenceof ε-sarcoglycan. This is in contrast to DGC preparations from Sgcb-nullmice in which ε-sarcoglycan was greatly reduced. Moreover, in Sgca-nullmice, some γ- and δ-sarcoglycan remained. In DGC preparations fromSgcb-null mice, however, all the sarcoglycans were observed to beabsent. Also, α-dystroglycan was absent from fractions 7-9, but remainedin the earlier fractions. Some β-dystroglycan also remained in Sgcb-nullmice. In summary, loss of β-sarcoglycan causes dissociation of thesarcoglycan and dystroglycan complex and also of ε-sarcoglycan whereasmice deficient for α-sarcoglycan show normal ε-sarcoglycan expression.These data indicate the presence of a ε-sarcoglycan containing complexin skeletal muscle. However, it is not associated with the tetramericunit of α-, β-, γ- and δ-sarcoglycan, since this complex is very muchreduced in Sgca-null mice, and ε-sarcoglycan is retained in these mice.Nevertheless, the expression of ε-sarcoglycan is obviously affected bythe β-sarcoglycan mutation, suggesting that β-sarcoglycan andε-sarcoglycan may be associated.

Methods of the Invention, Section II

[0069] Construction of Targeting Vector. Hind III fragments of a P1clone containing the mouse β-sarcoglycan gene (obtained from GenomeSystems, Inc.) were subcloned into pBluescript KS (+) (pBS) and analyzedusing restriction mapping and sequencing (Genbank/EMBL/DDBJ accessionnumber AF 169288). The long arm of homology in the targeting vector wasa 7.2 kb Hind III fragment upstream of exon 6, which had been subclonedinto pBS and cut with Xho1 to generate a 6.5 kb fragment. The short armwas a 1.8 Kpn1 fragment carrying approximately half of the intronbetween exons 2 and 3. These fragments were inserted into cloning sitesof pPNT flanking a PGK-neomycin resistance cassette to produce a DNAconstruct which contained 1800 bp of intron 2 and 6500 bp of sequenceswhich begin 498 directly downstream of exon 6, of the β-sarcoglycangene. The neomycin resistance gene was located between the intron 2sequences and the sequences downstream of exon 6. The vector included athymidine kinase cassette distal to the short arm. The mutant genetherefore lacked ˜7.5 kb which included exons 3, 4, 5 and 6, and introns3, 4, and 5.

[0070] Generation of Sgcb-Deficient Mice. The targeting vector waslinearized with Not1 and transferred into 2×10⁷ R1 ES cells byelectroporation (240 V, 500 μF; Bio-Rad Gene Pulser; Hercules, Calif.).Clones surviving growth in G418 and Gancyclovir were isolated. Targetingfidelity was determined by Southern blot analysis. Correct targetingresulted in a deletion of a region of approximately 7.5 kb, whichincluded 1606 bp of intron 2, the entire exon 3, 4, 5, 6, intron 3, 4,and 5, and also 498 bp immediately downstream of exon 6. The deletedregion was replaced with the PGK-neomycin cassette.

[0071] Cells from three correctly targeted clones were microinjectedinto C57BL/6J blastocysts and transferred into pseudopregnantrecipients. Chimeras from the three independently derived ES cells gaverise to heterozygous mice which in turn were mated to generatehomozygous mutants that were genotyped using Southern blot analysis onDNA from tail biopsies. All animals were kept in the animal care unit ofthe University of Iowa College of Medicine according to the animal careguidelines.

[0072] Northern Blot Analysis. Total RNA from control, heterozygous, andhomozygous-null mutant skeletal muscle was extracted using RNAzol B(Tel-Test, Friendswood, Tex.) according to manufacturer specifications.20 μg of total RNA was run on a 1.25% agarose gel containing 5%formaldehyde and transferred to Hybond N Membrane (Amersham Corp.,Arlington Heights, Ill.). RNA was cross-linked to the membrane using aStratagene UV cross-linker (La Jolla, Calif.). Membranes were thenprehybridized and hybridized with either a 203 bp exon 2 specific probe(Probe 1, FIG. 2) or a 253 bp exon 6 specific probe (Probe 2, FIG. 2).Washes were carried out at 65° C. in 1X SSC/1% SDS initially, then 0.1XSSC/1%SDS. Blots were exposed for autoradiography.

[0073] Histopathology Studies. Wild type, heterozygous and Sgcb-nullmice were anaesthetized with Metofane. Subsequently, the animals wereperfused with 15 ml of PBS followed by 15 ml of 10% buffered formalinfixative solution. After embedding the tissue in paraffin, hematoxylinand eosin (H&E) stained sections (4 μm) were prepared to characterizeskeletal and cardiac muscle pathology.

[0074] Evans Blue Dye Injection and Serum Creatine Kinase Analysis.Evans blue dye (EBD) (Sigma Chemical Co., St. Louis, Mo.) was dissolvedin PBS (10 mg/ml) and sterilized by passage through membrane filterswith a pore size of 0.2 μm. Mice were anaesthetized with Metofane andinjected in the retro-orbital sinus with 0.05 ml/10 g of body weight ofthe dye solution. Animals were sacrificed 4 hr after injection bycervical dislocation. Muscle sections for microscopic Evans bluedetection were incubated in ice-cold acetone at −20° C. for 10 min, andafter a rinse with PBS, sections were mounted in Vectashield mountingmedium (Vector Laboratories, Inc., Burlingame, Calif.) and observedunder a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Inc.,Thornwood, N.Y.). Quantitative, kinetic determination of creatine kinaseactivity in serum of wild-type, heterozygous and Sgcb-null mice wasmeasured using a Hitachi 917, on blood drawn from retro-orbital sinus.

[0075] Antibodies. Monoclonal antibody IIH6 against α-dystroglycan(Ervasti and Campbell, 1991) was previously characterized. Monoclonalantibodies Ad1/20A6 against α-sarcoglycan, βSarc/5B1 againstβ-sarcoglycan, and 35DAG/21B5 against γ-sarcoglycan were generated incollaboration with L. V. B. Anderson (Newcastle General Hospital,Newcastle upon Tyne, UK). Monoclonal antibody 43DAG/8D5 againstβ-dystroglycan was generated by L. V. B. Anderson (Newcastle GeneralHospital, Newcastle upon Tyne, UK). Rabbit polyclonal antibodies againstα-sarcoglycan (rabbit 98) (Roberds et al., J. Biol. Chem. 268:23739-23742 (1993)), δ-sarcoglycan (rabbits 215 and 229) (Holt et al.,Mol. Cell 1: 841-848 (1998)), ε-sarcoglycan (rabbit 232) (Duclos et al.,J. Cell Biol. 142: 1461-1471 (1998); Duclos et al., Neuromusc. Disord.8: 30-38 (1998)), sarcospan (rabbit 235) (Duclos et al., J. Cell Biol.142: 1461-1471 (1998); Duclos et al., Neuromusc. Disord. 8: 30-38(1998)), dystrophin (rabbit 31) (Ohlendieck et al., J. Cell Biol. 115:1685-1694 (1991)), the α2 subunit of dihydropyridine receptor (rabbit136) (Ohlendieck et al., J. Cell Biol. 115: 1685-1694 (1991)), and thelaminin α2 chain (Allamand et al., Hum. Mol. Gen. 6: 747-752 (1997))were described previously. The goat polyclonal antibody againstβ-sarcoglycan (goat 26) was also described previously (Duclos et al., J.Cell Biol. 142: 1461-1471 (1998); Duclos et al., Neuromusc. Disord. 8:30-38 (1998)). An affinity purified rabbit polyclonal antibody (rabbit245) was produced against a COOH-terminal fusion protein (amino acids167-291) of γ-sarcoglycan. In addition, an affinity purified rabbitpolyclonal antibody (rabbit 256) was produced against an NH₂-terminalfusion protein (amino acids 1-25) of sarcospan.

[0076] Microfil® Perfusion. Wild-type and Sgcb-null mice wereanesthetized with 75 mg/kg body weight Phenobarbital and a bilateralsternum incision was performed to expose the left atrium. 1 ml ofMicrofil®, a silicon rubber (Flow Tech., Carver, Mass.), was perfusedinto the left atrium. The heart continued beating for about 1 min andafter contraction stopped, the heart and diaphragm were rapidly excisedand cured on ice for about 10 min. Adequacy of vascular perfusion wasjudged by the white blush that developed in the ventricular wall as wellas a white filling of other main arteries including the mesentericartery and femoral artery. The hearts were fixed in 10% formalin for48-72 hr and cardiac tissue was sectioned into 2 mm thick transversecross sections. The diaphragms were taken out as whole tissue and werenot further cut. The tissues were subsequently cleared by sequential 24hr immersions in 25, 50, 75, 95 and finally 100% ethanol. On day 6,specimens were placed in pure methyl salicylate for 12-24 hr. All stepswere done at room temperature. Microvascular perfusion was visualizedwith trans-illumination and examined under low power magnification.

[0077] Immunofluorescence Analysis. For immunofluorescence analysis 7 μmtransverse cryosections were prepared from wild-type, heterozygous andSgcb-null mutant skeletal muscle, cardiac muscle, lung, bladder andesophagus. All following steps were performed at room temperature.Sections were blocked with 5% BSA in PBS for 20 min and then incubatedwith the primary antibodies for at least 90 min. After washing with PBS,sections were incubated with Cy3-conjugated secondary antibodies (1:200)for 1 hr and then washed in PBS. Subsequently, sections were mountedwith Vectashield (Vector Laboratories, Inc., Burlingame, Calif.)mounting medium and observed under a Zeiss Axioplan fluorescencemicroscope (Carl Zeiss Inc.) or an MRC-600 laser scanning confocalmicroscope (Bio-Rad Laboratories, Hercules, Calif.)

[0078] Immunoblot Analysis of Membrane Preparations. KCl-washedmembranes from skeletal and cardiac muscle and lung were prepared asdescribed previously (Ohlendieck et al., J. Cell Biol. 115: 1685-1694(1991)) with the addition of two protease inhibitors, calpeptin andcalpain inhibitor I (Duclos et al., J. Cell Biol. 142: 1461-1471 (1998);Duclos et al., Neuromusc. Disord. 8: 30-38 (1998)). Membranes wereresolved by SDS-PAGE on 3-12% linear gradient gels and transferred tonitrocellulose membranes. Immunoblot staining was performed aspreviously described (Ohlendieck et al., J. Cell Biol. 115: 1685-1694(1991)). Blots were also developed using enhanced chemiluminescence(SuperSignal, Pierce Chemical Co.).

[0079] Sucrose Gradient Fractionation of Skeletal MuscleDystrophin-Glycoprotein Complex. Skeletal muscle (1.5 g) was dissectedfrom wild-type, Sgca-null and Sgcb-null mice and snap frozen in liquidnitrogen. Frozen tissue was pulverized using a mortar and pestle cooledwith liquid nitrogen. The tissues were solubilized by douncehomogenization in 10 ml cold buffer A (50 mM Tris-HCl, pH 7.4, 500 mMNaCl, 1% digitonin) with a cocktail of protease inhibitors (0.6 μg/mlpepstatin A, 0.5 μg/ml aprotinin, 0.5 μg/ml leupetin, 0.1 mM PMSF, 0.75mM benzamidine, 5 μM calpain inhibitor I, and 5 μM calpeptin). Thehomogenate was rotated at 4° C. for 1 hr, and subsequently spun at142,400 g for 37 min at 4° C. The pellets were resolubilized with 5 mlbuffer A, rotated at 4° C. for 30 min, and centrifuged as before. Thetwo supernatants were pooled and incubated at 4° C. with WGA-Agarose(Vector Laboratories). The WGA-Agarose was washed extensively in bufferB (50 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.1% digitonin with the abovedescribed cocktail of protease inhibitors) and proteins were eluted with0.3 M N-acetyl glucosamine (Sigma Chemical Co.) in buffer B. Sampleswere concentrated to 0.3 ml, diluted 5-fold in buffer B, againconcentrated to 0.3 ml using Centricon 30-filters and applied to a 5-30%sucrose gradient at pH 7.4, as described previously (Ervasti et al., J.Biol. Chem. 266: 9161-9165 (1991)).

1. A mouse, and cells derived therefrom, homozygous for a disruptedδ-sarcoglycan gene, the disruption in said gene having been introducedinto the mouse or an ancestor of the mouse at an embryonic stage,wherein the disruption prevents the synthesis of functionalδ-sarcoglycan in cells of the mouse and results in the mouse having areduced amount of β- and ε-sarcoglycan and sarcospan and a disruption ofthe sarcoglycan-sarcospan complex in smooth muscle, and a reduced amountof sarcospan, α-, β-, γ-, and ε-sarcoglycan in the sarcolemma ofskeletal and cardiac muscles, compared to the amounts of said componentsin a mouse lacking disrupted δ-sarcoglycan genes.
 2. The mouse, andcells derived therefrom, of claim 1 wherein the disruption comprises adeletion of a region of 2992 base pairs, including 2277 base pairs ofintron 1, the entire exon 2, and 576 base pairs of intron 2, andreplacement of the deleted region with a PGK-neomycin cassette as amarker for neomycin resistance.
 3. The mouse, and cells derivedtherefrom, of claim 2 wherein the deletion results from theintroduction, into embryonic stem cells, of a DNA construct comprising:a) 5 kb of intron 1 and 5 kb of intron 2 of the δ-sarcoglycan gene; andb) a neomycin resistance gene inserted between intron 1 and intron 2 ofthe δ-sarcoglycan gene, the neomycin resistance gene being in theopposite transcriptional orientation as the δ-sarcoglycan exonsreplaced, wherein the construct lacks exon 2 of the δ-sarcoglycan gene.4. A mouse, and cells derived therefrom, homozygous for a disruptedβ-sarcoglycan gene, the disruption in said gene having been introducedinto the mouse or an ancestor of the mouse at an embryonic stage,wherein the disruption prevents the synthesis of functionalβ-sarcoglycan in cells of the mouse and results in the mouse having areduced amount of δ- and ε-sarcoglycan and sarcospan and α-dystroglycanin smooth muscle, and a disruption of the sarcoglycan-sarcospan complexin smooth muscle, and a reduced amount of sarcospan, α-, γ-, δ- andε-sarcoglycan in the sarcolemma of skeletal and cardiac muscles,compared to the amounts of the components in a mouse lacking disruptedβ-sarcoglycan genes.
 5. The mouse, and cells derived therefrom, of claim4 wherein the disruption comprises a deletion of a region of about 7.5kb, including 1606 bp of intron 2, the entirety of exon 3, exon 4, exon5, exon 6, intron 3, intron 4, and intron 5, and 498 bp immediatelydownstream of exon 6, and replacement of the deleted region with aPGK-neomycin cassette as a marker for neomycin resistance.
 6. The mouse,and cells derived therefrom, of claim 5 wherein said disruption resultsfrom the introduction, into embryonic stem cells, of a DNA constructcomprising: a) 1800 bp of intron 2 and 6500 bp of sequences which begin498 bp directly downstream of exon 6, of the β-sarcoglycan gene; and b)a neomycin resistance gene inserted between intron 2 and the sequencesdownstream of exon 6 of the β-sarcoglycan gene, wherein said constructlacks exon 3, exon 4, exon 5, exon 6, intron 3, intron 4, and intron 5,of the β-sarcoglycan gene.
 7. A method for treating mammalian autosomalrecessive limb-girdle muscular dystrophy type 2F in an individual,comprising the steps: a) providing an expression vector which encodes awild-type form of δ-sarcoglycan; and b) introducing the expressionvector into skeletal and smooth muscle tissue of the individual underconditions appropriate for expression of the wild-type form ofδ-sarcoglycan in said tissues.
 8. The method of claim 7 wherein theexpression vector is selected from the group consisting of an adenovirusexpression vector, a gutted adenovirus expression vector and anadeno-associated expression vector.
 9. The method of claim 7 where theexpression vector contains a muscle tissue-specific promoter.
 10. Themethod of claim 7 wherein the expression vector is introduced intoskeletal muscle by intramuscular injection.
 11. A method for treatingmammalian autosomal recessive limb-girdle muscular dystrophy type 2E inan individual, comprising the steps: a) providing an expression vectorwhich encodes a wild-type form of β-sarcoglycan; and b) introducing theexpression vector into skeletal and smooth muscle tissue of theindividual under conditions appropriate for expression of the wild-typeform of β-sarcoglycan in said tissues.
 12. The method of claim 11wherein the expression vector is selected from the group consisting ofan adenovirus expression vector, a gutted adenovirus expression vectorand an adeno-associated expression vector.
 13. The method of claim 11where the expression vector contains a muscle tissue-specific promoter.14. The method of claim 11 wherein the expression vector is introducedinto skeletal muscle by intramuscular injection.
 15. A method foridentifying a therapeutic compound useful for treatment of an individualdiagnosed with δ-sarcoglycan-deficient limb-girdle muscular dystrophy,comprising: a) providing a mouse homozygous for a disruptedδ-sarcoglycan gene; b) administering a candidate compound to the mouseof step a); and c) assaying for therapeutic effects on the mouse of stepa), the detection of therapeutic effects being an indication that theadministered compound is a therapeutic compound for treatment of theindividual.
 16. The method of claim 15 wherein the candidate compound isadministered to the mouse by means to contact the candidate compoundwith smooth muscle cells of the mouse.
 17. The method of claim 15wherein the candidate compound is administered to the mouse by means tocontact the candidate compound with skeletal muscle cells of the mouse.18. The method of claim 15 wherein the candidate therapeutic compound isadministered to the mouse by means to contact the candidate compoundwith cardiac muscle cells of the mouse.
 19. The method of claim 15wherein the candidate compound is a gene and the gene is administeredunder conditions appropriate for expression of the gene in cells of themouse.
 20. A method for identifying a therapeutic compound useful fortreatment of an individual diagnosed with β-sarcoglycan-deficientlimb-girdle muscular dystrophy, comprising: a) providing a mousehomozygous for a disrupted β-sarcoglycan gene; b) administering acandidate compound to the mouse of step a); and c) assaying fortherapeutic effects on the mouse of step a), the detection oftherapeutic effects being an indication that the administered compoundis a therapeutic compound for treatment of the individual.
 21. Themethod of claim 20 wherein the candidate compound is administered to themouse by means to contact the candidate compound with smooth musclecells of the mouse.
 22. The method of claim 20 wherein the candidatecompound is administered to the mouse by means to contact the candidatecompound with skeletal muscle cells of the mouse.
 23. The method ofclaim 20 wherein the candidate therapeutic compound is administered tothe mouse by means to contact the candidate compound with cardiac musclecells of the mouse.
 24. The method of claim 20 wherein the candidatecompound is a gene and the gene is administered under conditionsappropriate for expression of the gene in cells of the mouse.
 25. Atherapeutic method for treating ischemic heart disease caused by reducedexpression of the sarcoglycan-sarcospan complex in vascular smoothmuscle cells of an individual, comprising contacting the vascular smoothmuscle cells of the individual with a vascular smooth muscle relaxant.26. The therapeutic method of claim 25 wherein the vascular smoothmuscle relaxant is Nicorandil.
 27. The method of claim 25 wherein thereduced expression of the sarcoglycan-sarcospan complex in vascularsmooth muscle cells of the individual is due to a defect in theδ-sarcoglycan genes of the individual.
 28. The method of claim 25wherein the reduced expression of the sarcoglycan-sarcospan complex inthe vascular smooth muscle cells of the individual is due to a defect inthe β-sarcoglycan genes of the individual.
 29. A method for preventingischemic injury in skeletal and cardiac muscle of an individual causedby reduced expression of the sarcoglycan-sarcospan complex in thevascular smooth muscle cells of the individual, the method comprisingcontacting the vascular smooth muscle cells of the individual with avascular smooth muscle relaxant.
 30. The method of claim 29 wherein thevascular smooth muscle relaxant is Nicorandil.
 31. A method for treatingmammalian autosomal recessive limb-girdle muscular dystrophy type 2F inan individual, comprising administering a vascular smooth musclerelaxant to the individual.
 32. A method for treating mammalianautosomal recessive limb-girdle muscular dystrophy type 2E in anindividual, comprising administering a vascular smooth muscle relaxantto the individual.
 33. A method for identifying a therapeutic compoundfor the treatment of ischemic heart disease in an individual caused byreduced expression of the sarcoglycan-sarcospan complex in the vascularsmooth muscle cells of the individual, comprising: a) providing a mousewhich has reduced expression of the sarcoglycan-sarcospan complex in thevascular smooth muscle cells; b) administering a candidate compound tothe mouse of step a) by means to contact the candidate compound with thevascular smooth muscle cells of the mouse; and c) assaying fortherapeutic effects on the mouse of step a), the detection oftherapeutic effects being an indication that the administered compoundis a therapeutic compound.
 34. The method of claim 33 wherein the mouseis homozygous for a disrupted δ-sarcoglycan gene.
 35. The method ofclaim 33 wherein the mouse is homozygous for a disrupted β-sarcoglycangene.
 36. A method for identifying a therapeutic compound for theprevention of ischemic injury in skeletal and cardiac muscle of anindividual which is caused by reduced expression of thesarcoglycan-sarcospan complex in vascular smooth muscle cells of theindividual, the method comprising: a) providing a mouse which hasreduced expression of the sarcoglycan-sarcospan complex in vascularsmooth muscle cells; b) administering a candidate compound to the mouseof step a) by means to contact the vascular smooth muscle cells of themouse; and c) assaying for protection of the mouse which has receivedthe candidate compound from ischemic injury, the determination ofprotection being an indication that the administered compound is atherapeutic compound.
 37. The method of claim 36 wherein the mouse ishomozygous for a disrupted δ-sarcoglycan gene.
 38. The method of claim36 wherein the mouse is homozygous for a disrupted β-sarcoglycan gene.